The oocyte plays an integral role in regulating folliculogenesis within the mammalian ovary. In particular, the oocyte has been shown to act on granulosa cells to regulate follicle formation perinatally, stimulate granulosa cell proliferation, modulate granulosa cell gene expression, and influence steroidogenesis (Reviewed in (Eppig, J. J., Dev. Biol., 5:51-59 (1994)). The granulosa cells in the pre-ovulatory follicle can be separated into two populations with regard to their proximity to the oocyte; cumulus granulosa cells closely surround the oocyte while the mural granulosa cells are located around the periphery of the follicle separated from the oocyte by an antrum. Cumulus cells secrete a hyaluronic acid-rich matrix during cumulus expansion and are extruded with the oocyte during ovulation. This expanded matrix is a critical factor for reproductive integrity since it binds the oocyte and cumulus cells together, facilitates follicular extrusion and oviductal fimbria capture, and allows sperm penetration and fertilization (Salustri, A., et al., Zygote, 4:313-315 (1996)). On the other hand, mural granulosa cells synthesize proteases important for follicle rupture at ovulation, remain within the ovary after the cumulus cell-oocyte complex is released, and eventually undergo terminal differentiation to form the corpus luteum. These positional and functional differences in the granulosa cell populations suggests that gradients of oocyte-secreted factors modulate gene expression and eventual cell differentiation. In vitro studies demonstrate that oocyte-secreted growth factors regulate granulosa cell synthesis of hyaluronic acid, urokinase plasminogen activator (uPA) and LH receptor as well as steroidogenesis and luteinization (Salustri, A., et al., Zygote 4:313-315 (1996); Vanderhyden, B. C., et al., Endocrinology, 133:423-426 (1993); Eppig, J. J., et al., Biol. Reprod., 56:976-984 (1997); Eppig, J. J., et al., Human Reprod., 12:127-132 (1997); Nekola, M. V. and Nalbandov, A. V., Biol. Reprod., 4:154-160 (1971); El-Fouly, M. A., et al., Endocrinology, 87:288-293 (1970)). However, the identities of the oocyte-derived factors that regulate these somatic cell functions remain largely unknown.
Mouse growth differentiation factor 9 (mGDF-9) is expressed in the ovary and specifically in oocytes (PCT WO94/15966). A GDF-9 knockout mouse demonstrated female infertility due to an early block in folliculogenesis (Dong et al., Nature, 383:531-535 (1996)), however, the GDF-9 receptor and the cell type expressing the GDF-9 receptor have not been identified. Discovery of the cell type expressing the GDF-9 receptor would assist in defining the role of GDF-9 in fertility, and would provide an assay system for identifying agents which target GDF-9 activity.
Described herein is an in vitro assay for growth differentiation factor 9 (GDF-9) based upon the discovery that GDF-9 binds to granulosa cells found in the ovary of mammals. As also described herein, expression of particular proteins are enhanced and/or inhibited upon binding of GDF-9 to the receptors on the granulosa cells.
The present invention relates to a method of identifying an agent which alters (modulates) activity of GDF-9. As used herein the term xe2x80x9caltersxe2x80x9d refers to partial and/or complete inhibited activity (decreased activity) or enhanced activity (increased activity). The method involves combining cells having receptors for GDF-9 (e.g., granulosa cells) and a gene (one or more) whose expression is regulated by binding of GDF-9 to the receptors (e.g., hyaluronan synthase, steroidogenic acute regulatory protein (StAR), luetinizing hormone (LH) receptor, cyclooxygenase 2 (COX-2), urokinase plasminogen activator (uPA), kit ligand, activin/inhibin xcex2B and follistatin); GDF-9; and an agent to be assessed (test sample). The combination produced in the test sample is maintained under conditions appropriate for binding of GDF-9 to the receptors on the cells. The extent to which expression of the gene occurs is then determined, wherein alteration of expression of the gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates that the agent alters GDF-9 activity. The method can further comprise a control step wherein the extent to which binding occurs in the presence of the agent is compared to the extent to which binding occurs in the absence of the agent. For example, the extent to which expression of the gene occurs in the test sample is compared to the extent to which expression of the gene occurs in a control sample (e.g., a combination comprising the cells having receptors for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptors, and GDF-9).
The present invention also relates to a method of identifying an agent which is an inhibitor of GDF-9 activity. In one embodiment, granulosa cells; GDF-9; and an agent to be assessed are combined. The combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells; and the extent to which expression of a gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined. Inhibition of expression of the gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates the agent inhibits GDF-9 activity. In another embodiment, granulosa cells; GDF-9; and an agent to be assessed are combined. The combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells; and the extent to which expression of uPA, a gene regulated by binding of GDF-9 to the receptors, occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined. Increased expression of the uPA gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates that the agent inhibits GDF-9 activity.
The present invention also relates to a method of identifying an agent which is an enhancer of GDF-9 activity. In one embodiment, granulosa cells; GDF-9; and an agent to be assessed are combined. The combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells and the extent to which expression of a gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined. Enhanced expression of the gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed is indicative of an agent which is an enhancer of GDF-9 activity. In another embodiment, granulosa cells; GDF-9; and an agent to be assessed are combined. The combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells; and the extent to which expression of a uPA gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined. Decreased expression of the uPA gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates that the agent enhances GDF-9 activity.
The methods of the present invention can also be used to identify an agent which inhibits fertility in a mammal (e.g., human). Alternatively, the methods of the present invention can be used to identify an agent which enhances fertility in a mammal.
The present invention also relates to a method of identifying an agent which is an agonist of GDF-9. In this method, cells having receptors for GDF-9 and a gene, wherein expression of the gene is regulated by binding of GDF-9 to the receptor; GDF-9; and an agent to be assessed are combined. The combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the cells, and the extent to which expression of the gene regulated by binding of GDF-9 to the receptors occurs is determined, wherein expression of the gene in the presence of the agent to be assessed indicates that the agent is an agonist of GDF-9.
In addition, the present invention relates to a method of identifying an agent which is an agonist or antagonist of GDF-9. In this method, cells having receptors for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptor; GDF-9 and an agent to be assessed are combined. The combination produced is maintained under conditions appropriate for binding of the agent to receptors on the cells. The extent to which binding of the agent to the receptors on the cells occurs is determined, wherein binding of the agent to the GDF-9 receptors indicates that the agent is an agonist or an antagonist of GDF-9.
The extent to which binding of GDF-9 to the receptor on the cells occurs can be determined in a variety of ways. For example, the extent to which binding occurs is determined by directly measuring a gene product (e.g., nucleic acids such as DNA, RNA of the gene; protein, peptide encoded by the gene) of the gene which is regulated by binding of GDF-9 to the receptors. In one embodiment, the gene encodes a protein involved in the synthesis of hyaluronic acid (e.g., hyaluronan synthase), and the extent to which binding of GDF-9 to the receptors on the granulosa cells occurs is determined by measuring the production of a product of the gene (e.g., RNA coding for hyaluronan synthase), wherein an increase in production the gene product indicates the agent is an enhancer of GDF-9 activity and a decrease of production of gene product indicates the agent is an inhibitor of GDF-9 activity. In another embodiment, the gene encodes a protein involved in the synthesis of progesterone (e.g., StAR), and the extent to which binding of GDF-9 to the receptors on the granulosa cells occurs is determined by measuring the production of a product of the gene (e.g., RNA coding for StAR), wherein an increase in the production of the gene product indicates that the agent is an enhancer of GDF-9 activity, and a decrease in production of the gene product indicates that the agent is an inhibitor of GDF-9 activity. In yet another embodiment, the gene encodes a protein involved in the production of plasmin (e.g., uPA), and the extent to which expression of the gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors is determined by measuring the production of a product of the gene (e.g., RNA coding for uPA), wherein a decrease in the expression of the gene product indicates the agent is an enhancer of GDF-9 activity, and an increase in expression of the gene product indicates the agent is an inhibitor of GDF-9 activity. The extent to which binding of GDF-9 to the receptor on the cells occurs can also be determined by measuring a product or function (e.g., hyaluronic acid, progesterone and/or plasmin) attributed to the activity of proteins encoded by the gene whose expression is regulated by binding of GDF-9 to the receptor on the cell.
The findings that granulosa cells respond to GDF-9 and that this response can be determined by means of one or more inducible genes allows for an in vitro bioassay for GDF-9. Such an assay can be used to diagnose fertility problems in mammals, and identify inhibitors, enhancers, antagonists and analogues of GDF-9 which can be used, for example, to diagnose and/or treat fertility problems in mammals.